Muscle atrophy can result from several different causes: undernutrition, non-use of the muscles (for example immobilization following a fracture), cancer or other serious disease (heart or kidney failure) inducing cachexia, or resulting naturally from the aging of individuals (sarcopenia). This atrophy can result from a reduction in protein synthesis and/or from an increase in proteolysis and, as appropriate, is accompanied by fibrosis and/or by infiltration by adipose tissue. The identification of the factors and mechanisms controlling muscle protein synthesis and muscle proteolysis thus represents a prerequisite for designing appropriate treatments for these pathological conditions.
FIG. 1, which is part of the prior art, shows the principal pathways of protein synthesis and proteolysis in muscles (reconstructed according to Zhao et al., 2008 and Little et al., 2009).
Muscle protein synthesis is essential, and is essentially controlled at the translational level. It requires of course an adequate nutritional intake of amino acids. It is stimulated by physical activity and regulated by numerous factors, at the forefront of which are IGF-1 and androgens (Little et al., 2009).
TABLE 1factors and molecules which act on proteinsynthesis and proteolysis in musclesProtein synthesisProteolysisFactorStimulationInhibitionStimulationInhibitionExercise+(+)Denervation−Fasting, anorexia−+Amino acids+−Insulin+−GH/IGF-1+−FGF++Vitamin D+Adrenaline+−Acetylcholine−Ocytocine+Apelin+Testosterone+−Estradiol+−Triiodothyronine+(T3)Myostatin−+TGFβ−+Follistatin−Angiotensin II−+Angiotensin-(1-7)−Glucocorticoids−+PIF−+IL-1β+IL-6(−)+TNF-α, IFN-γ−+Anti-inflammatories−
Myofibril proteolysis is performed via the proteasome, while the mitochondria are destroyed by autophagy (Zhao et al., 2008). Satellite cell apoptosis mechanisms are also described (Murphy et al., 2010).
Myostatin, produced in an autocrine manner by the muscles themselves, represents a particularly important factor, since it acts both by stimulating proteolysis and by inhibiting protein synthesis. It also stimulates fibrosis (Li et al., 2008).
Aging is accompanied by a modification of the various regulatory factors (Walston et al., 2012): physical activity is often reduced, protein/vitamin nutrition may be insufficient and, following meals, the contents of circulating amino acids, an increase of which is required to stimulate protein synthesis, show a reduced increase that may be due to splanchnic sequestration (Boirie et al., 1997). Moreover, aging is accompanied by considerable hormonal modifications: an increase in myostatin (Leger et al., 2008), a reduction in androgens (Seidman, 2007) and in growth hormone (Macell et al., 2001; Sattler, 2013), and also an increase in inflammation markers (IL-6, TNF-α etc., Schaap et al., 2009; Verghese et al., 2011), will in particular be noted. These various modifications are unfavorable for protein synthesis, whereas these promote proteolysis, hence the gradual reduction in muscle size (sarcopenia). They also cause a modification in the distribution of muscle fiber types to the detriment of the fast fibers, which is reflected by a decrease in muscle strength (dynapenia). Finally, the development of connective tissue within the muscles (fibrosis) is witnessed.
In an obesity context, the situation is worsened for several additional reasons: fat infiltration of the muscles worsens the inflammatory context, insulin resistance reduces the effect of IGF-1 on protein synthesis, without considering that mobility is reduced by the excess weight (Stenholm et al., 2009).
FIG. 2, which is part of the prior art, illustrates the worsening of sarcopenia in an obesity context (according to Quillot et al., 2013).
In any event, in the absence of treatment, sarcopenia is a process which can only get worse, until total loss of mobility. However, sarcopenia is not the only process which results in skeletal muscle atrophy. Atrophy also occurs during immobilization (for example following a fracture), during prolonged fasting (or a slimming diet), or during serious pathological conditions (for example cancers, AIDS) which cause cachexia. Mention may also be made of various muscle dystrophies of genetic origin. These various situations have a certain number of characteristics in common with sarcopenia, but with a respective weight different than the triggering factors (Tisdale, 2007; Saini et al., 2009).
Known Possible Treatments
Various methods for preventing/treating sarcopenia have thus been envisaged and tested. They are first and foremost physical exercise, the effectiveness of which is established (Bonnefoy et al, 2000; Bonnefoy, 2008; Ryan et al., 2013). Thus, following exercise carried out over a period of 8 weeks, increases in muscle strength of 180% and in muscle mass of 11% have been observed (Fiatarone et al., 1990). However, optimal effectiveness would require several hours of physical exercise per day, which is difficult to envision over long periods of time.
An increased intake of protein synthesis substrates, whether by giving rapidly digestive proteins according to an optimized timing (Coëffier et al., 2009; Aussel et al., 2013), and also a supplement of certain amino acids or their metabolites (leucine, HMB [β-hydroxy-ρ-methylbutyrate], citrulline, ornithine), can increase muscle protein synthesis (Li & Heber, 2011).
Various pharmaceutical treatments aim to correct the modifications of the hormonal context associated with aging (Crenn, 2013). They comprise:                sex hormones such as testosterone (White et al., 2013) or variants thereof, SARMs (Selective Androgen Receptor Modulators), or non-sex hormones such as growth hormone (Liu et al., 2003) and IGF-1, ghrelin or progranulin, or even vitamin D;        myostatin inhibitors (antibodies directed against the molecule or its receptor, or myostatin precursor peptide) (Murphy et al., 2010; Han & Mitch, 2011);        molecules which target the renin-angiotensin system, such as inhibitors of ACE or angiotensin 1-7 (Dalla Libera et al., 2001; Shiuchi et al., 2004; Kalupahana & Moustaid-Moussa, 2012; Allen et al., 2013);        β-adrenergic receptor agonists (Ryall et al., 2004, 2007);        varied natural substances, or even more complex extracts of plant origin (for example, isoflavones: Aubertin-Leheudre et al., 2007; olive oil extract: Pierno et al., 2014; resveratrol: Shadfar et al., 2011; Bennett et al., 2013).        
The great diversity of these treatments attests to the difficulty of treating a multifactorial pathological condition, the triggering factors of which have not been totally identified. Furthermore, several candidate molecules have side effects (in the case of sex hormones, SARMs or β-agonists, for example), or have as yet been studied only on animal models. All these elements explain the lack of available medicaments on the market.
To date, research studies target more particularly myostatin, by inhibiting its action with, for example, anti-myostatin antibodies or anti-receptor antibodies (Dumonceaux et al., 2010; Greenberg, 2012; Sakuma & Yamaguchi, 2012; Arounleut et al., 2013; Buehring & Binkley, 2013; Collins-Hooper et al., 2014; White & Le Brasseur, 2014).
Phytoecdysones, and more particularly the 20-hydroxyecdysone (20E), have been the subject of numerous pharmacological studies, which began in Japan and then in Uzbekistan, and have subsequently developed in various other countries.
These studies have revealed the antidiabetic and anabolic properties of this molecule. Its stimulating effects on protein syntheses in muscles are observed in rats in vivo (Syrov, 2000; Tóth et al., 2008; Lawrence, 2012) and on C2C12 murine myotubes in vitro (Gorelick-Feldman et al., 2008). It is an effect at the level of translation, which involves the phosphorylation of the p70S6K ribosomal protein, at the end of a cascade involving the Akt/PkB protein kinase, a pathway also used by IGF-1 to stimulate protein synthesis.
Using the same C2C12 cells, Zubeldia et al. (2012) have moreover shown that an Ajuga turkestanica extract enriched with phytoecdysones (20-hydroxyecdysone and turkesterone) inhibits the transcription of myostatin and of caspase 3 (a protein involved in apoptosis processes).
Moreover, 20-hydroxyecdysone has antifibrotic properties, which have not been demonstrated on muscles, but in the kidneys, where the fibrosis mechanisms take place very similarly (Hung et al., 2012). It thus opposes the effects of TGFβ, a protein similar to myostatin, and in particular the stimulation of Smad 2,3 caused by this substance. It can thus be considered that 20-hydroxyecdysone could have similar effects on muscles (or the heart).
20-Hydroxyecdysone reduces body fat in mice fed with a fat-enriched diet (Kizelsztein et al., 2009; Foucault et al., 2012) or in ovariectomized female rats, a model of menopause (Seidlova-Wuttke et al., 2010).
Some of the effects described above in animal models have been found in clinical studies, which are even fewer in number. Thus, 20-hydroxyecdysone increases physical capacity (Azizov et al., 1995; Gadhzieva et al., 1995) and muscle mass (Simakin et al., 1988) and causes a loss of abdominal fat mass in obese and overweight volunteers (Wuttke et al., 2013; Foucault et al., 2014; PCT patent application WO 2013/068704).
However, 20E and the metabolites thereof have poor bioavailability in mice (Dzhukharova et al., 1987; Hikino et al., 1972), in rats (Kapur et al., 2010 and Seidlova-Wuttke et al., 2010) and in humans (Brandt 2003; Bolduc, 2006). Their overall performance is among other things not entirely satisfactory in relation to muscle quality improvement applications.
Several studies have shown that turkesterone (11α,20-dihydroxyecdysone), a metabolite derived from 20E, shows a greater activity than that of 20E in vivo (Syrov et al., 2001: Bathori et al., 2008). There is still today, for therapeutic applications targeting an improvement in muscle quality both in obese mammals and in sarcopenic mammals, a need for novel compounds which have good bioavailability, expressed more particularly in terms of high plasma exposure coefficient, while at the same time having an overall activity greater than that of 20E on muscle quality improvement, this overall activity being expressed in terms of performance relating to inhibition of myostatin gene expression combined with increased protein synthesis in the mammal.